Zinc-chelating ratiometric fluorescent probes and related methods

ABSTRACT

Benzoxazole fluorescent sensor compounds and related ratiometric imaging methods for zinc metal ion.

RELATED METHODS

This application claims priority benefit from provisional patentapplication No. 60/485,587 filed Jul. 8, 2003, the entirety of which isincorporated herein by reference.

The United States government has certain rights to this inventionpursuant to Grant Nos. DK52627 and GM38784 from the National Institutesof Health and the National Science Foundation, respectively, toNorthwestern University.

BACKGROUND OF THE INVENTION

Zinc is an essential element in both enzymatic and biological systems,and is physiologically the second most abundant transition metal. Theinorganic physiology of intracellular zinc is poorly understood but ofemerging importance in understanding a variety of human disorders anddisease states. Histochemical studies of mammalian tissues including theprostate, the insulin secreting beta cells of pancreatic islets, and thedentate neurons of the hippocampus reveal patterns of Zn(II)accumulation that are disrupted in some types of prostatic cancer,diabetes and neurodegerative disorders respectively. The function ofzinc in these tissues or even within compartments of single cellorganisms such as S. cerevease remains controversial.

In order to investigate the functions of such spectroscopically silentmetal ions (e.g., Ca²⁺ and Zn⁺²) in biological systems, fluorescentsensor molecules that respond to a specific metal ion in the excitationor emission spectrum have shown to be useful tools. Several kinds offluorescence probes for Zn²⁺ that can be used under physiologicalcondition have been reported to date, most of these utilize “on-off”fluorescent signaling system, in which fluorescence intensity increaseslineally upon increasing Zn²⁺ concentration. In these cases, however,the determination of the accurate Zn²⁺ concentration in the cells shouldbe impossible because the fluorescence intensity depends on many factorssuch as the cell thickness, incubation time, illumination intensity, dyeconcentration, and the photobleach of dye itself.

Confocal fluorescence microscopy has proved to be a central tool inunderstanding calcium biology and has the potential to resolve theseissues in zinc biology. On the other hand, two-photon excitation (TPE)fluorescence microscopy provides significant advantages over standardlaser confocal approaches by providing deeper sectioning, lessphototoxicity and selective excitation of a smaller focal volume (i.e.,femptoliter), thus decreasing background fluorescence. Such advances ininstrumentation could be applied to the study of zinc physiology butwould require the parallel development of new zinc-specific chemicalprobes that operate within cells.

Ratiometric probes that exhibit a large shift in the excitation and/oremission spectrum upon binding with the cation can minimize experimentalerror as the fluorescence intensity ratio between the apo form and theZn²⁺-bound form is dependent on only free Zn²⁺ concentration. Recently,two ratiometric probes for Zn²⁺ have been reported; however, asexcitation probes, two different excitation wavelengths are needed forratiometic cell imaging. As a result, this technique is not suitable fora confocal laser microscopy.

Such development has been an ongoing concern in the art. Protein-basedzinc probes are useful in a variety of physiological experiments, butcannot be used without microinjection into each cell. Severalbenzofuran-based and coumazin probes have been used but only forexcitation ratio imaging of a zinc loaded cell. Others in the art used2-(2′-hydroxyphenyl)benzoxazole (HBO) as a fluorophore, which exhibitsdual emission, utilizing ESIPT (Excited State Intramolecular ProtonTransfer); however, intracellular analysis/imaging was not described.The search continues for a class of emission ratiometric probes forintracellular zinc, especially those with utility in two-photonfluorescence microscopy of mammalian cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: (A) UV-vis spectral change observed upon the additionof Zn²⁺ (0.5 μM each) to 5 μM of Zinbo-5 in 50 mM HEPES (pH 7.20, 0.1 MKNO₃) containing 5% DMSO. (B) Absorbance at 337 nm and 376 nm is plottedtoward [Zn²⁺]_(total)/[Zinbo-5].

FIG. 2. (A) Emission spectra of Zinbo-5 with the excitation at 356 nm inZn²⁺/EGTA buffered system (50 mM HEPES, pH 7.20, 0.1 M KNO₃; 10 mM EGTA,1–9 mM zinc sulfate) at 0, 0.14, 0.29, 0.66, 1.1, 1.8, 2.6, 4.0, 6.1,and 11 nM free Zn²⁺, respectively. (B) Plots of the fluorescenceintensity ratio between 395 nm and 443 nm (I₄₄₃/I₃₉₅) with a best curvefor a dissociation constant of 2.1±0.1×10⁻⁹.

FIG. 3. UV determination of dissociation constant, K_(d): (A) Absorptionratio between at 340 nm and at 374 nm (Abs₃₇₄/Abs₃₄₀) in Zn²⁺-bufferedsolution (50 mM HEPES, pH 7.20, 0.1 M KNO₃; 10 nM EGTA, 1–9 mM zincsulfate).

FIG. 4. Metal ion selectivity of Zinbo-5. Bars are a presentation of thefluorescence ratio I₄₄₃/I₃₉₅. Heavy metals (20 μM) and Na⁺, K⁺, Ca²⁺,Mg²⁺ (5 mM) were added to 1.5 μM Zinbo-5 in the presence of 10 μM EDTA.

FIG. 5. Emission ratio images of fibroblast (LTK) cells treated withZinbo-5. (A) Brightfield transmission image; (B) ratio image of theintensities at 445 nm and 402 nm; (C) ratio image following a 30 minutetreatment with 10 μM zinc sulfate and 20 μM pyrithione at pH 7.4, 25°C., followed by wash with Zinbo-5 stock; (D) ratio image of the samefield after a 15 minute treatment with 1 mM TPEN.

FIG. 6. Structural formulae of compounds and intermediates en routethereto, in accordance with this invention.

FIG. 7. A schematic representation of the synthesis of the compounds andintermediates represented by the structural formulae of FIG. 6.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide a range of fluorescent sensor probe compounds and/or methodsrelating to ratiometric imaging of zinc ion, thereby overcoming variousdeficiencies and shortcomings of the prior art, including those outlinedabove. It will be understood by those skilled in the art that one ormore aspects of this invention can meet certain objectives, while one ormore other aspects can meet certain other objectives. Each objective maynot apply equally, in all its respects, to every aspect of thisinvention. As such, the following objects can be viewed in thealternative with respect to any one aspect of this invention.

It is an object of the present invention to provide one or morebenzoxazole compounds for use in ratiometric imaging applications, andto also quantitatively measure or assay zinc metal ion concentrationsand changes thereof.

It is another object of the present invention to provide one or morebenzoxazole compounds of the sort described herein substituted at aposition on the fused ring to enhance zinc interaction, solventsolubility, cellular uptake and/or quantum yield.

It is another object of the present invention to provide a fluorescentsensor compound having a dissociation constant for zinc in the manomolarrange, thereby allowing real time study of zinc physiology in livingcells.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and descriptions of variousembodiments, and will be readily apparent to those skilled in the arthaving knowledge of various zinc probes and analytic/imaging techniques.Such objects, features, benefits and advantages will be apparent fromthe above as taken into conjunction with the accompanying examples,data, figures and all reasonable inferences to be drawn therefrom.

Accordingly, in part, the present invention includes a compound of aformula

wherein R₁ and R₂ are independently selected from hydrogen, hydroxy,alkyl and alkoxy; and X₁ is (2-pyridinylalkyl); and X₂ is selected fromhydrogen, alkyl, alkylhydroxy and (2-pyridinylalkyl). In certainembodiments, X₂ can be but is not limited to hydrogen, alkyl and(2-pyridinylalkyl) or as can be otherwise varied as would be understoodby those skilled in the art to affect solvent solubility and/or cellularuptake. As described herein, such compounds can chelate, bind, complexor otherwise interact with zinc metal ions. As such, compounds of thepresent invention can be employed with a number of pharmaceutical and/orbiological applications, including but not limited to zinc assays andzinc chelation in the treatment of various disease states.

Accordingly, the present invention also includes a compositioncomprising a Zn⁺² metal ion and one of the aforementioned compounds, butwithout limitation, a compound of a formula

wherein R₁ and R₂ are as provided elsewhere herein. While the zinc ionsmay be found in an extracellular environment, such compositionscomprising intracellular zinc ion can find application in one of theaforementioned assays or disease treatments.

In addition, such compounds can be used in conjunction with one or moremethods for ratiometric imaging of intracellular Zn⁺² metal ion. Such amethod comprises (1) providing a compound of the type described herein;(2) treating a mammalian cell with such a compound; and (3) irradiatingthe cell, then comparing fluorescent emissions of the compound unboundand bound with zinc. As would be understood by those skilled in the art,such imaging can be accomplished with a two-photon excitation laser, butcan also be provided using various other spectroscopic techniques.

Likewise, a further departure from the prior art, the present inventionalso provides a method of using a polydentate benzoxazole compound toassay or measure Zn⁺² metal ion. Such a method comprises (1) providing apolydentate benzoxazole compound of the sort described herein,substituted at the 4-position of fused ring system; and (2) irradiatingthe compound in the presence of the metal ion, whether intracellular orextracellular, and comparing a ratio of fluorescence emissionintensities. Plotting such a ratio against a standard concentrationcurve can be used to determine zinc concentrations and/or changesthereto, whether intracellular or extracellular.

One such fluorescent sensor compound—referred to as Zinbo-5,herein—illustrates various aspects of this invention: it is cellpermeable, binds free Zn²⁺ with a K_(d) in the nanomolar range, andshows significant zinc-induced changes in quantum yield and in both theexcitation and emission maxima. Compounds of this invention, such asZinbo-5, are well suited for two-photon emission ratio microscopy andreadily reveal changes in intracellular zinc within single cells. Suchcompounds and related methods can be applied to real time studies ofzinc physiology in living cells.

The compounds of this invention can be characterized by a highlyfluorescent benzoxazole core substituted with a variety of Zn-chelatinggroups pendent thereto. The synthesis of one such compound, Zinbo-5,which employs the aminomethyl pyridine moiety, involves reaction of aMOM-protected phenol aldehyde derivative with amino-m-cresol andoxidation of the product with barium manganate to provide thebenzoxazole derivative. Bromination of the methyl group using NBS,coupling with 2-aminomethyl pyridine, and deprotection by p-toluenesulfonic acid yielded the compound referred herein as Zinbo-5. (A moredetailed synthesis is provided in the following examples.)

Other such benzoxazole compounds, with a range of substitutedhydroxyphenyl moieties and/or various substitutions pendent to theheterocyclic core, can be prepared using synthetic procedures of thetype described herein or straightforward modifications thereof—as neededto effect the desired structural variation(s).

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compounds, compositions and/or methods ofthe present invention, including the use of the benzoxazole sensors ofthis invention, as are available through the synthetic methods describedherein. In comparison with the prior art, the present compounds,compositions and/or methods provide results and data which aresurprising, unexpected and contrary thereto. While the utility of thisinvention is illustrated through the use of several compounds andimaging techniques, it will be understood by those skilled in the artthat comparable results are obtainable with various other compounds andmethodologies, as are commensurate with the scope of this invention.

Example 1a Synthesis of Zinbo-5

With reference to Scheme 1, below, the following material and reagentswere used in preparation of compound Zinbo-5:2,4,5-trimethoxybenzaldehyde (Aldrich, 98%), boron trichloride (Aldrich,1.0 M solution in CH₂Cl₂), chloromethyl methyl ether (Aldrich, tech),2-amino-m-cresol (Aldrich, 96%), N-bromosuccinimide (Aldrich, 99%),2-(aminomethyl)pyridine (Aldrich, 99%). NMR: (δ in ppm vs SiMe₄ (0 ppm,¹H, 400 MHz or 500 MHz), CDCl₃ (77.0 ppm, ¹³C, 500 MHz). Columnchromatography: Merck silica gel (70–230 mesh). TLC: 0.25 mm, Mercksilica gel 60 F254, visualizing at 254 mm.

Exhibit 1b 4,5-dimethoxy-2-hydroxybenzaldehyde (2)

A solution of boron trichloride (50 mL, 1.0 M solution in CH₂Cl₂) wasslowly added to a solution of 2,4,5-trimethoxybenzaldehyde (3.92 g, 20mmol) in CH₂Cl₂ (200 mL) at −78° C. (dry ice/acetone bath). The mixturewas warmed to room temperature and stirred for three hours. 10 mL of HCl(37%) aqueous solution was poured into the resulting solution at 0° C.and extracted with CH₂Cl₂ (150 mL×3). The combined organic layer waswashed with saturated aqueous NaCl (200 mL×2), water (100 mL×1), driedover MgSO₄, and evaporated to afford 3.72 g of a light brown solid (3.72g, 99%): ¹H-NMR (400 MHz, CDCl₃) δ3.88 (s, 3H), 3.94 (s, 3H), 6.47 (s,1H), 6.91 (s, 1H), 9.70 (s, 1H).

Example 1c 4,5-dimethoxy-2-methoxymethoxybenzaldehyde (3)

Chloromethyl methyl ether (1.61 g, 20 mmol) at 0° C. was added to asolution of 1 (1.75 g, 14.9 mmol) and diisopropylethylamine (3.5 mL, 20mmol) in CH₂Cl₂ (30 mL) was added, and the mixture was stirred for 15hours at room temperature. Then water was added to the solution and theaqueous layer was extracted with CH₂Cl₂ (100 mL×3). The combined organiclayer was washed with saturated aqueous NaCl (100 mL×2) followed bywater (100 mL×1), dried over MgSO₄, and evaporated to afford 3.56 g ofbrown oil (2.56 g, 76%): ¹H-NMR (500 MHz, CDCl₃) δ3.55 (s, 3H), 3.89 (s,3H), 3.95 (s, 3H), 5.26 (s, 2H), 6.77 (s, 1H), 7.32 (s, 1H), 10.35 (s,1H).

Example 1d 2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole(5)

A solution of 3 (2.26 g, 10 mmol) and 2-amino-m-cresol (1.23 g, 10 mmol)in benzene (70 mL) was refluxed for 15 hours using an additional funnelto remove water. The reaction mixture was cooled to room temperature andthe solvent was removed in vacuo to afford the Schiff base compound 4 asa dark orange solid, which was used for the next reaction withoutfurther purification.

A solution of 4 (3.31 g, 10 mmol) and BaMnO₄ (10.3 g, 40 mmol) inbenzene (70 mL) was refluxed for 5 hours under a flow of dry N₂ gas.After the reaction mixture was cooled to room temperature, BaMnO₄ wasremoved through Celite and the filtrate was concentrated in vacuo. Theblack residue was purified by silica gel column chromatography (CHCl₃)to afford 2.07 g of a pale yellow solid, 5 (2.07 g, 62%): ¹H-NMR (400MHz, CDCl₃) δ2.66 (s, 3H), 3.58 (s, 3H), 3.95 (s, 3H), 3.97 (s, 3H),5.28 (s, 2H), 6.84 (s, 1H), 7.12 (d, 1H, J=8.0 Hz), 7.21 (t, 1H, J=8.0Hz), 7.39 (d, 1H, J=8.0 Hz), 7.63 (s, 1H).

Example 1e4-bromomethyl-2-(4,5-dimethoxy-2-methoxymethoxyphenyl)benzoxazole (6)

A mixture of 5 (1.39 g, 4.23 mmol), N-bromosuccinimide (0.75 g, 4.23mmol) and AIBN (33 mg, 0.2 mmol) in CCl₄ (70 mL) was refluxed for 15hours under a flow of dry N₂ gas. The reaction mixture was cooled to 0°C. and the precipitate was removed by filtration while maintaining thetemperature at 0° C. After the solvent was evaporated, the residue waswashed with small amount of ethanol several times to afford a pinkishsolid 6 (1.14 g, 66%): ¹H-NMR (500 MHz, CDCl₃) δ3.60 (s, 3H), 3.97 (s,3H), 3.99 (s, 3H), 4.96 (s, 2H), 5.31 (s, 2H), 6.85 (s, 1H), 7.30 (t,1H, J=8.0 Hz), 7.40 (d, 1H, J=8.0 Hz), 7.51 (d, 1H, J=8.0 Hz), 7.66 (s,1H).

Example 1f 2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethyl benzoxazole (7)

A mixture of 6 (612 mg, 1.5 mmol), 2-(aminomethyl)pyridine (1.08 g, 10mmol), and Na₂CO₃ (excess) in CH₃CN (30 mL) was stirred overnight atroom temperature. Insoluble material was removed by filtration and thefiltrate was concentrated in vacuo. Water (20 mL) was added to theresidue, which was extracted with ethyl acetate (20 mL) three times. Thecombined organic layer was washed with brine and water, dried overMgSO₄, and evaporated. The resulting oily material was purified bysilica gel column chromatography (CHCl₃-ethyl acetate) to afford 7 aspale yellow oil (112 mg, 17%): ¹H-NMR (500 MHz, CDCl₃) δ53.56 (s, 3H),3.95 (s, 3H), 3.97 (s, 3H), 4.02 (s, 2H), 4.30 (s, 2H), 5.27 (s, 2H),6.85 (s, 1H), 7.15 (dd, 1H, J=7.5, 5.0 Hz), 7.28 (t, 1H, J=7.5 Hz), 7.33(d, 1H, J=7.5 Hz), 7.42 (d, 1H, J=7.5 Hz), 7.47 (d, 1H, J=7.5 Hz), 7.64(m, 2H), 8.55 (d, 1H, J=5.0 Hz).

Example 1g 2-(4,5-dimethoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole (Zinbo-5)

A mixture of 7 (100 mg, 0.23 mmol) and p-toluenesulfonic acidmonohydrate (190 mg, 1 mmol) in methanol (20 mL) was stirred overnightat room temperature. After the solvent was evaporated, ethyl acetate (20mL) was added to the resulting residue which was neutralized by Na₂CO₃,washed with brine and water, dried over MgSO₄, and concentrated in vacuoto give a pale yellow solid that was purified by silica gel columnchromatography (ethyl acetate) and crystallized from ethanol to afford awhite solid of Zinbo-5 (80 mg, 89%): ¹H-NMR (500 MHz, CDCl₃) δ3.95 (s,3H), 3.96 (s, 3H), 4.01 (s, 2H), 4.21 (s, 2H), 6.66 (s, 1H), 7.17 (dd,1H, J=7.5, 5.0 Hz), 7.31 (t, 1H, J=8.0 Hz), 7.37–7.40 (m, 2H), 7.41 (s,1H), 7.48 (d, 1H, J=8.0 Hz),7.67 (t, 1H, J=7.5 Hz), 8.66 (d, 2H, J=5.0Hz), 11.27 (s, 1H); Anal. Calcd for C₂₂H₂₁N₃O₄: C, 67.51; H, 5.41; N,10.74. Found: C, 67.12; H, 5.25; N, 10.57.

(a) BBr₃ in CH₂Cl₂, rt (b) CH₂OCH₂Cl, TEA in CH₂Cl₂, rt (c)amino-m-cresol in benzene, reflux (d) BaMnO₄ in benzene, reflux (e) NBS,AIBN in CCl₄, reflux (f) 2-aminomethylpyridine derivative, K₂CO₃ inCH₃CN, rt (g) TsOH in CH₃OH, rt.

Example 2

UV Visible and Fluorescence Spectroscopy

The UV absorption spectra of Zinbo-5 were recorded on a Hewlett-Packard8453 spectrometer. Fluorescence spectra were recorded using a PTIfluorimeter (Photon Technology International). To reduce fluctuations ofthe excitation intensity during measurement, the lamp was turned on for1 hour prior to the experiment. The path length was 1 cm with a cellvolume of 3.0 μL. Quantum yields were determined to be 0.02 for the apoform and 0.10 for the zinc complex using fluorescein in 0.1 N NaOH(Φ=0.95).

Example 3a

Binding Constant Determination

A series of HEPES (2-[4-(2-hydoroxyethyl)-1-piperazinyl]ethanesulfonicacid) buffer solutions (50 mM, pH 7.20, 0.1 M KNO₃) containing variousamounts of ZnSO₄ (0˜9.5 mM) and 10 mM of EGTA(ethylenebis(oxyethylenenitrilo) tetraacetic acid) were prepared. Theconcentration of free Zn²⁺ was calculated with [EGTA]_(total),[Zn²⁺]_(total), and K′_(Zn-EGTA), the apparent binding constant at agiven pH and ionic strength. K′_(Zn-EGTA) value was calculated from Eq.1 at pH=7.20K′_(Zn-EGTA)=K_((ZnL))(1+10^((pK) ^(LMH) ^(−pH))/((1+10^((pH−pK) ^(Zn)⁾)(1+10^((pK) ¹ ^(−pH))10^((pK) ¹ ^(+pK) ² ^(−2pH)))  (Eq 1)using the following published pK and log K values for EGTA; pK₁=9.40,pK₂=8.79, pK₃=2.70, pK₃=2.70, pK_(LMH)=9.40, log K(ZnL)=12.6 (25° C.,μ=0.1 M).² All protonation constants were corrected upward by 0.11 whenworking in 0.1 M ionic strength. Thus, K′_(Zn-EGTA) value at pH 7.20,0.1 M ionic strength is 3.80×10⁸ M⁻¹.The calculated [ZN²⁺]free concentration of each solution is:

[Zn²⁺]_(total) (mM) 0.5 1 2 3 4 5 6 7 8 9 [Zn²⁺]_(free) (nM) 0.14 0.290.66 1.1 1.8 2.6 4.0 6.1 11 24

The ratio between 395 nm and 443 nm intensities in the emission spectrumof each solution was measured with the excitation wavelength at 356 nmand was fitted to the following equation (Eq. 2).R=(R_(min)K_(d)+R_(max)[Zn²⁺])/(K_(d)+[Zn²⁺])  (Eq. 2)

Example 3b

A mixture of aqueous buffer (50 mM HEPES, pH 7.20, 0.1 M KNO₃) and DMSO(5%, v/v) was used for the UV titration of Zinbo-5 with Zn²⁺. Asignificant decrease in the absorption band of the apo at 337 nm and anincrease of a new band at 376 nm was observed with a distinct isosbesticpoint at 356 nm (FIG. 1A). The absorption bands at 337 nm and 376 nmlinearly decreased and increased, respectively, up to a 1:1[Zn²⁺]/[Zinbo-5] ratio, indicating formation of a 1:1 complex (FIG. 1B).

Example 3c

FIG. 2A shows the emission spectra of Zinbo-5 when excited at 356 nm,which is the isosbestic point of the absorption titration spectrum atvarious free Zn²⁺ concentrations. The apo form exhibits a characteristicband at 407 nm that shifts to 443 nm upon binding with Zn²⁺. Comparisonswithin the Zinbo family indicate that the 4-hydroxyl function gives riseto a larger shift relative to the unsubsituted forms (data not shown).In contrast to those model compounds which exhibit a blue shift inemission upon zinc binding, Zn²⁺ binding to Zinbo-5 leads to a red shiftin the emission, suggesting a different photophysical mechanism for thepolydentate complexes of this invention.

Example 3d

The apparent dissociation constant (K_(d)=2.2±0.1 nM at pH 7.20) forZn²⁺ was determined by plotting (FIG. 2B) the fluorescence intensityratio between 395 nm and 443 nm (or UV absorbance as shown in FIG. 3)against log[Zn²⁺]_(free) and fitting this data as described in theliterature. The corresponding pZn value (−log[Zn²⁺], where [Zn²⁺] is thefree metal concentration calculated for an aqueous solution containing10 mM ligand and 1 mM zinc ion at pH 7.20 and 0.1 M ionic strength) is9.3. Such results are comparable to the affinity of prior art Zinquin,Zinpyr and ZnAF probes (Table 1), below, which have been used in studiesof zinc-loaded cells. The Zn²⁺ affinity of Zinbo-5 is much higher thanthat of di(2-picolyl)amine (DPA), indicating—without limitation—that thephenolate oxygen and benoxazole nitrogen are likely chelating the metalas the third and fourth ligands.

TABLE 1 Calculated pZn Values (−log[Zn]_(free)) for a SolutionContaining 10 μM of the Indicated Ligand, 1 μM of Zn(II) at pH 7.20,0.1M Ionic Strength, and 25° C. Ligand pZn Ligand pZn NTA 9.0 TPEN 16.0EGTA 9.5 Zinquin acid 9.3 EDTA 14.3 carbonic anhydrase 12.4Zinpyr-1^((a)) 10.1 Zinpyr-2^((b)) 10.3 Zinpyr-4^((c)) 10.1 ZnAF-1^((d))10.1 ZnAF-2^((d)) 9.5_(—) ZnAF-R1^((e)) 10.1 ZnAF-R2^((e)) 9.5_(—)L^(3(f)) 10.8 Zinbo-5 9.3 ^((a))Walkup, G. K.; Burdette, S. C.; Lippard,S. J.; Tsien, R. Y. J Am Chem Soc 2000, 122, 5644–5645. ^((b))Burdette,S. C.; Walkup, G. K.; Spingler, B.; Tsien, R. Y.; Lippard, S. J. J AChem Soc 2001, 123, 7831–7841. ^((c))Burdette, S. C.; Frederickson, C.J.; Bu, W., and Lippard, S. J. J Am Chem Soc 2003, 125, 1778–1787.^((d))Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J AmChem Soc 2000, 122, 12399–12400. ^((e))Maruyama, S.; Kikuchi, K.;Hirano, T.; Urano, Y.; Nagano, T. J Am Chem Soc 2002, 124, 10650–10651.^((f))Koike, T.; Watanabe, T.; Aoki, S.; Kimura, E.; Shiro, M. J Am ChemSoc. 1996, 118, 12696–12703.

Example 4

A survey of Zinbo-5 fluorescence in a series of biological bufferscontaining physiologically relevant metal ions reveals that only Zn²⁺and Cd²⁺ induced an emission shift as shown in FIG. 2A, whereas otherheavy metal ions, Mn²⁺, Co²⁺, Ni²⁺, Fe²⁺ and Cu²⁺, quenched thefluorescence (FIG. 4). Na⁺, K⁺, Ca²⁺ and Mg²⁺ afforded no fluorescentresponse even at metal concentrations as high as 5 mM. Neither high norlow concentrations of alkali metal or alkaline earth metal ions alteredthe fluorescent response of Zinbo-5 to Zn²⁺, suggesting that this probemay be useful in a wide range of biological and microscopicapplications.

Example 5

Ratio Imaging Methods

The Zinbo-5 probe was tested in the mouse fibroblast LTK cell line.Cells were fixed, treated with a saturated solution of Zinbo-5 and thenimaged with two-photon excitation (TPE) laser scanning microscopy usingon Zeiss 510 LSM (upright configuration). The excitation beam producedby the femtosecond pulsed Ti:sapphire laser (Tsunami, Spectra-Physics; 8W Millennia pump) was tuned to 710 nm, (pumping power 6.5 W with 0.5 Wentering the Zeiss AOM) was passed through an LSM 510 microscope with anHFT 650 dichroic (Zeiss) and focused onto coverslip adherent fibroblastsusing a 63×oil-immersion objective (Zeiss). The NLO META scan head(Zeiss) allowed data collection in 10.7 nm windows centered at 445 nmand 402 nm.

Cells were imaged in the presence of Zinbo-5 to provide the contrast ofunbound dye outside of the cells (FIG. 5). After imaging, the same fieldof cells was treated with 10 μM zinc sulfate and 20 μM pyrithione, azinc-selective ionophore, resulting in an anticipated increase in theintracellular ratio of emissions from 445 nm and 402 nm (FIG. 5, d–f).Finally, the intracellular ratio was made equal to that of thesurrounding unbound dye by treating the same field of view with TPEN(FIG. 5, g–i). These data indicate that Zinbo-5 may be an excellentchoice for a ratiometric zinc sensor in biological studies.

Example 6

Cell Culture

LTK cells were grown as previously published (Nasir, M. S., et al., JBIC(1999) 4:775–783). Cells grown on coverslips were fixed with 4%formaldehyde on ice for 20 minutes and washed with cold PBS beforestaining. The cells were stained on ice for 30 minutes in 2 mL of PBSand 4 μL of a 5 mM solution of Zinbo-5 in DMSO, thus in a saturatedsolution with a final Zinbo-5 concentration limited by the maximal watersolubility of 1.5 μM. Coverslips were attached to slides usingSecure-Seal spacers (Molecular Probes) with the saturated stainingsolution in the spacer well.

Example 7

Microscopy and Ratio Imaging

512×512 pixel images were obtained in the presence of Zinbo-5 using aZeiss LSM 510 set to a frame scan speed of four and an average of four.META detection and Zeiss software (LSM 510, version 3.0; Carl Zeiss,Inc.) in lambda mode allowed detection and data collection in two 10.7nm channels centered at 402 and 445 nm. Images collected in these twochannels were evenly divided and then multiplied by 30 to improvecontrast using the Zeiss software ratio function. Images were collectedwith a Plan-Apo 63×oil objective, 1.4 n.a. (Carl Zeiss, Inc.) at 25° C.(See FIG. 5A)

For cells grown in standard conditions, the ratio image suggests verylow levels of available Zn²⁺ (FIG. 5B). The ratio image changes in amanner dependent upon the availability of Zn(II) within the cell: theratio is clearly higher when the intracellular Zn(II) is increased byaddition of 10 μM zinc sulfate and 20 μM pyrithione, a zinc-chelatingionophore (FIG. 5C). This corresponds to an average intracellularincrease in the ratio of ca. 15%. Next, the signals in these ratioimages were shown to originate from Zn²⁺/Zinbo-5 complex by treatmentwith a tighter binding competitor. Treatment of cells with an excess ofthe cell-permeable, high affinity zinc chelator, N,N,N′N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), decreases the intracellularratio by 30% to equal that of the unbound probe (FIG. 5D). This andcontrol experiments on untreated cells indicate that autofluorescencedoes not contribute to the ratio images under these conditions.

The emission ratio imaging studies and data of the preceding examplesindicate that Zinbo-5 and other compounds and/or methods of thisinvention can readily reveal changes in intracellular or extracellularzinc availability. The photophysical data further suggest that thecompounds of this invention can also be used in standard laser scanningconfocal imaging or in epifluorescence excitation ratio imagingapproaches to studying zinc in tissues and cells. Such methods areparticularly amenable to investigation of live tissue samples in realtime and are being applied to studies of the hippocampus, wherephysiological fluctuations in synaptic zinc concentration are estimatedto be as high as 100–300 μM or as low 2 nM.⁵

In accordance with this invention, various other fluorescent probecompounds are available and can be used as described herein, as would beunderstood by those skilled in the art made aware of this invention.Without limitation, several such compounds and their respectivesynthetic intermediates (F1, F2, etc.) are prepared as described below,with reference to the structural formulae and schematics of FIGS. 6 and7.

Example 8 Production of 5-methoxy-2-methoxymethoxybenzaldehyde (F1)

To a solution of 2-hydroxy-5-methoxybenzaldehyde (10 g, 67.5 mmol) anddiisopropylethylamine (14 mL, 80 mmol) in CH₂Cl₂ (100 mL) was addedchloromethyl methyl ether (6.1 mL, 80 mmol) at 0° C., and the mixturewas stirred for 15 hours at room temperature. Then, water was added tothe solution, the aqueous layer was extracted with CH₂Cl₂ (100 mL×3).The combined organic layer was washed with saturated aqueous NaCl (100mL×2), water (100 mL×1), dried over MgSO₄, and evaporated to afford theobjective compound as a brown oil (11.76 g, 91%), which was used fornext reaction without further purification: ¹H-NMR (400 MHz, CDCl₃)δ3.52 (s, 3H), 3.81 (s, 3H), 5.24 (s, 2H), 7.12 (dd, 1H, J=9.6, 3.2 Hz),7.18 (d, 1H, J=9.6 Hz), 7.32 (d, 1H, J=3.2 Hz), 10.47 (s, 1H).

Example 9 Production of 4,5-dimethoxy-2-methoxymethoxybenzaldehyde (F2)

4,5-dimethoxy-2-methoxymethoxybenzaldehyde (F2) was prepared in asimilar manner for the preparation of5-methoxy-2-methoxymethoxybenzaldehyde (F1) by using4,5-dimethoxy-2-hydroxybenzaldehyde (1.75 g, 14.9 mmol) instead of2-hydroxy-5-methoxybenzaldehyde in 76% (2.56 g): ¹H-NMR (500 MHz, CDCl₃)δ3.55 (s, 3H), 3.89 (s, 3H), 3.95 (s, 3H), 5.26 (s, 2H), 6.77 (s, 1H),7.32 (s, 1H) 10.35 (s, 1H).

Example 10 Production of 4-methoxymethoxybenzaldehyde (F3)

4-methoxymethoxybenzaldehyde (F3) was prepared in a similar manner forthe preparation of 5-methoxy-2-methoxymethoxybenzaldehyde (F1) by using4-hydroxybenzaldehyde (4.48 g, 40 mmol) instead of2-hydroxy-5-methoxybenzaldehyde in 96% (6.40 g): ¹H-NMR (500 MHz, CDCl₃)δ3.53 (s, 1H), 5.24 (s, 2H), 7.17 (d, 1H, J=9.0 Hz), 7.35 (d, 1H, J=9.0Hz), 10.52 (s, 1H).

Example 11 Production of2-[(5-Methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4)

A solution of 5-methoxy-2-methoxymethoxybenzaldehyde (F1) (2.77 g, 16.5mmol) and 2-amino-m-cresol (2.03 g, 16.5 mmol) in benzene (70 mL) wasrefluxed for 15 h using an additional funnel to remove water. After thereaction mixture was cooled to room temperature, the solvent was removedin vacuo and the resulting residue was washed with ethanol to afford theobjective compound as a orange solid (84%), which was used for nextreaction without further purification: ¹H-NMR 400 MHz, DMSO-d₆) δ2.31(s, 3H), 3.45 (s, 3H), 3.85 (s, 3H), 5.18 (s, 2H), 6.59 (s, 1H), 6.77(d, 1H, J=8.0 Hz), 6.83 (d, 1H, J=8.0 Hz), 6.99 (t, 1H, J=8.0 Hz), 7.03(dd, 1H, J=9.0, 2.5 Hz), 7.14 (d, 1H, J=9.0 Hz), 7.68 (s, 1H), 8.95 (s,1H).

Example 12 Production of2-[(4,5-dimethoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F5)

2-[(4,5-dimethoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F5)was prepared in a similar manner for the preparation of2-[(5-Methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4) byusing 4,5-dimethoxy-2-methoxymethoxybenzaldehyde (F2) (2.26 g, 10 mmol)instead of 5-methoxy-2-methoxymethoxybenzaldehyde (F1) in 86% (2.80 g):¹H-NMR (500 MHz, CDCl₃) δ3.53 (s, 3H), 5.24 (s, 3H), 7.17 (d, 1H, J=9.0Hz), 7.35 (d, 1H, J=9.0 Hz), 10.52 (s, 1H).

Example 13 Production of2-[(4-methoxymethoxybenzylidene)amino]-3-methylphenol (F6)

2-[(4-methoxymethoxybenzylidene)amino]-3-methylphenol (F6) was preparedin a similar manner for the preparation of2-[(5-Methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4) byusing 4-methoxymethoxybenzaldehyde (F3) (3.32 g, 20 mmol) instead of5-methoxy-2-methoxymethoxybenzaldehyde (F1) in 84% (4.56 g): ¹H-NMR (500MHz, CDCl₃) δ3.53 (s, 3H), 5.24 (s, 3H), 7.17 (d, 1H, J=9.0 Hz), 7.35(d, 1H, J=9.0 Hz), 10.52 (s, 1H).

Example 14 Production of2-[(2,5-dimethoxybenzylidene)amino]-3-methylphenol (F7)

2-[(2,5-dimethoxybenzylidene)amino]-3-methylphenol (F7) was prepared ina similar manner for the preparation of2-[(5-Methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4) byusing 2,5-dimethoxybenzaldehyde (3.32 g, 20 mmol) instead of5-methoxy-2-methoxymethoxybenzaldehyde (F1) in 82% (4.44 g): ¹H-NMR (400MHz, CDCl₃) δ2.32 (s, 3H), 3.84 (s, 6H), 6.77 (d, 1H, J=7.6 Hz), 6.82(d, 1H, J=2.0 Hz), 6.83 (d, 1H, J=7.6 Hz), 6.91 (d, 1H, J=9.2 Hz), 6.99(t, 1H, J=7.6 Hz), 7.06 (dd 1H, J=9.2, 2.0 Hz), 7.69 (s, 1H), 8.94 (s,1H).

Example 15 Production of2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F8)

A solution of2-[(5-methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4)(1.51 g, 5 mmol) and BaMnO₄ (5.13 g, 20 mmol) in benzene (50 mL) wasrefluxed for 5 hours under dry N₂ gas flowing. After the reactionmixture was cooled to room temperature, BaMnO₄ was removed throughCelite and the filtrate was concentrated in vacuo. The black residue waspurified by silica gel column chromatography (CHCl₃) to afford theobjective compound as a pale yellow soild (976 mg, 65%): ¹H-NMR (500MHz, CDCl₃) δ2.68 (s, 3H), 3.55 (s, 3H), 3.87 (s, 3H), 5.25 (s, 2H),7.02 (dd, 1H, J=9.0, 3.0 Hz), 7.15 (d, 1H, J=8.0 Hz), 7.21 (d, 1H, J=9.0Hz), 7.24 (t, 1H, J=8.0 Hz), 7.42 (d, 1H, J=8.0 Hz), 7.63 (d, 1H, J=3.0Hz).

Example 16 Production of2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F9)

2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F9) wasprepared in a similar manner for the preparation of2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F8) by using2-[(4-methoxymethoxybenzylidene)amino]-3-methylphenol (F6) (3.31 g, 10mmol) instead of2-[(5-methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4) in62% (2.07 g): ¹H-NMR (400 MHz, CDCl₃) δ2.66 (s, 3H), 3.58 (s, 3H), 3.95(s, 3H), 3.97 (s, 3H), 5.28 (s, 2H), 6.84 (s, 1H), 7.12 (d, 1H, J=8.0Hz), 7.21 (t, 1H, J=8.0 Hz), 7.39 (d, 1H, J=8.0 Hz), 7.63 (s, 1H).

Example 17 Production of 2-(2,5-dimethoxyphenyl)-4-methylbenzoxazole(F10)

2-(2,5-dimethoxyphenyl)-4-methylbenzoxazole (F10) was prepared in asimilar manner for the preparation of2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F8) by using2-[(2,5-dimethoxybenzylidene)amino]-3-methylphenol (F7) (5.43 g, 20mmol) instead of2-[(5-methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4) in85% (4.55 g): ¹H-NMR (500 MHz, CDCl₃) δ3.53 (s, 3H), 5.24 (s, 3H), 7.17(d, H, J=9.0 Hz), 7.35 (d, 1H, J=9.0 Hz), 10.52 (s, 1H).

Example 18 Production of 2-(4-methoxymethoxyphenyl)-4-methylbenzoxazole(F11)

2-(4-methoxymethoxyphenyl)-4-methylbenzoxazole (F11) was prepared in asimilar manner for the preparation of2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F8) by using2-[(4-methoxymethoxybenzylidene)amino]-3-methylphenol (F7) (5.43 g, 20mmol) instead of2-[(5-methoxy-2-methoxymethoxybenzylidene)amino]-3-methylphenol (F4) in92% (4.92 g): ¹H-NMR (500 MHz, CDCl₃) δ3.53 (s, 3H), 5.24 (s, 3H), 7.17(d, 1H, J=9.0 Hz), 7.35 (d, 1H, J=9.0 Hz), 10.52 (s, 1H).

Example 19 Production of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12)

A mixture of 2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole(F8) (1.78 g, 5.95 mmol), N-bromosuccinimide (1.01 g, 5.95 mmol), andAIBN (82 mg, 0.5 mmol) in CCl₄ (100 mL) was refluxed for 15 h under dryN₂ gas flowing. The reaction mixture was cooled to 0° C., theprecipitation was removed by filtration maintaining the temperature.After the solvent was evaporated, the residue was washed with smallamount of ethanol several times to afford a pinkish solid (1.72 g, 76%).¹H-NMR (500 MHz, CDCl₃) δ3.57 (s, 3H), 3.88 (s, 3H), 4.96 (s, 2H), 5.27(s, 2H), 7.05 (dd, 1H, J=8.5, 3.0 Hz), 7.22 (d, 1H, J=8.5 Hz), 7.34 (t,1H, J=8.0 Hz), 7.42 (d, 1H, J=8.0 Hz), 7.54 (d, 1H, J=8.0 Hz), 7.66 (d,1H, J=3.0 Hz).

Example 20 Production of4-bromomethyl-2-(4,5-dimethoxy-2-methoxymethoxyphenyl)benzoxazole (F13)

4-bromomethyl-2-(4,5-dimethoxy-2-methoxymethoxyphenyl)benzoxazole (F13)was prepared in a similar manner for the preparation of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) byusing 2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F9)(1.39 g, 4.23 mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F8) in 66%(1.14 g): ¹H-NMR (500 MHz, CDCl₃) δ3.60 (s, 3H), 3.97 (s, 3H), 3.99 (s,3H), 4.96 (s, 2H), 5.31 (s, 2H), 6.85 (s, 1H), 7.30 (t, 1H, J=8.0 Hz),7.40 (d, 1H, J=8.0 Hz), 7.51 (d, 1H, J=8.0 Hz), 7.66 (s, 1H).

Example 21 Production of4-bromomethyl-2-(2,5-dimethoxyphenyl)benzoxazole (F14)

4-bromomethyl-2-(2,5-dimethoxyphenyl)benzoxazole (F14) was prepared in asimilar manner for the preparation of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) byusing 2-(2,5-dimethoxyphenyl)-4-methylbenzoxazole (F10) (4.55 g, 16.9mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F8) in 56%(3.30 g): ¹H-NMR (500 MHz, CDCl₃) δ3.60 (s, 3H), 3.97 (s, 3H), 3.99 (s,3H), 4.96 (s, 2H), 5.31 (s, 2H), 6.85 (s, 1H), 7.30 (t, 1H, J=8.0 Hz),7.40 (d, 1H, J=8.0 Hz), 7.51 (d, 1H, J=8.0 Hz), 7.66 (s, 1H).

Example 22 Production of4-bromomethyl-2-(4-methoxymethoxyphenyl)benzoxazole (F15)

4-bromomethyl-2-(4-methoxymethoxyphenyl)benzoxazole (F15) was preparedin a similar manner for the preparation of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) byusing 2-(4-methoxymethoxyphenyl)-4-methylbenzoxazole (F11) (4.04 g, 15mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-methylbenzoxazole (F8) in 80%(4.16 g): ¹H-NMR (500 MHz, CDCl₃) δ3.60 (s, 3H), 3.97 (s, 3H), 3.99 (s,3H), 4.96 (s, 2H), 5.31 (s, 2H), 6.85 (s, 1H), 7.30 (t, 1H, J=8.0 Hz),7.40 (d, 1H, J=8.0 Hz), 7.51 (d, 1H, J=8.0 Hz), 7.66 (s, 1H).

Example 23 Production of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F16)

A mixture of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) (567mg, 1.5 mmol), 2-(aminomethyl)pyridine (1.08 g, 10 mmol), and Na₂CO₃(excess) in CH₃CN (30 mL) was stirred overnight at room temperature.After filtration of the reaction mixture, the filtrate was concentratedin vacuo. Water (20 mL) was added to the residue, which was extractedwith CHCl₃ (20 mL) three times. The combined organic layer was washedwith brine and water, dried over MgSO₄, and evaporated. The resultingoily material was purified by silica gel column chromatography(CHCl₃-ethyl acetate) to afford the objective compound as a pale yellowoil (386 mg, 63%). ¹H-NMR (400 MHz, CDCl₃) δ3.52 (s, 3H), 3.87 (s, 3H),4.01 (s, 2H), 4.30 (s, 2H), 5.23 (s, 2H), 7.02 (dd, 1H, J=9.2, 1.2 Hz),7.15 (dd, 1H, J=7.6, 4.4 Hz), 7.21 (d, 1H, J=9.2 Hz), 7.29–7.36 (m, 2H),7.41 (d, 1H, J=7.6 Hz), 7.49 (d, 1H, J=7.6 Hz), 7.62–7.65 (m, 2H), 8.55(d, 1H, J=4.4 Hz).

Example 24 Production of2-(5-methoxy-2-methoxymethoxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F17)

A mixture of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) (567mg, 1.5 mmol), N-methyl-N-(2-pyridylmethyl)amine (183 mg, 1.5 mmol), andNa₂CO₃ (excess) in CH₃CN (30 mL) was stirred overnight at roomtemperature. After filtration of the reaction mixture, the filtrate wasconcentrated in vacuo. Water (20 mL) was added to the residue, which wasextracted with CHCl₃ (20 mL) three times. The combined organic layer waswashed with brine and water, dried over MgSO₄, and evaporated. Theresulting oily material was purified by silica gel column chromatography(CHCl₃-ethyl acetate) to afford the objective compound as a pale yellowoil (386 mg, 63%). ¹H-NMR (400 MHz, CDCl₃) δ3.52 (s, 3H), 3.87 (s, 3H),4.01 (s, 2H), 4.30 (s, 2H), 5.23 (s, 2H), 7.02 (dd, 1H, J=9.2, 1.2 Hz),7.15 (dd, 1H, J=7.6, 4.4 Hz), 7.21 (d, 1H, J=9.2 Hz), 7.29–7.36 (m, 2H),7.41 (d, 1H, J=7.6 Hz), 7.49 (d, 1H, J=7.6 Hz), 7.62–7.65 (m, 2H), 8.55(d, 1H, J=4.4 Hz).

Example 25 Production of4-bis(2-pyridylmethyl)aminomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole(F18)

4-bis(2-pyridylmethyl)aminomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)-benzoxazole(F18) was prepared in a similar manner for the preparation of2-(5-methoxy-2-methoxymethoxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F17) using di(2-picolyl)amine (317 mg, 1.59 mmol) instead ofN-methyl-N-(2-pyridylmethyl)amine in 92% (730 mg): ¹H-NMR (500 MHz,CDCl₃) δ3.53 (s, 3H), 3.87 (s, 3H), 3.94 (s, 4H), 4.23 (s, 2H), 5.24 (s,2H), 7.03 (dd, 1H, J=9.0, 3.5 Hz), 7.12 (dd, 1H, J=7.5, 5.0 Hz), 7.22(d, 1H, J=9.0 Hz), 7.34 (t, 1H, J=7.5 Hz), 7.48 (d. 1H, J=7.5 Hz), 7.56(d, 1H, J=7.5 Hz), 7.64–7.68 (m, 3H), 7.75 (d, 2H, J=7.5 Hz), 8.52 (d,2H, J=5.0 Hz).

Example 26 Production of2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F19)

2-(4,5-dimethoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethyl-benzoxazole(F19) was prepared in a similar manner for the preparation of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F16) using4-bromomethyl-2-(4,5-dimethoxy-2-methoxymethoxyphenyl)benzoxazole (F13)(612 mg, 1.59 mmol) instead of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) in17% (112 mg); ¹H-NMR (500 MHz, CDCl₃) δ3.56 (s, 3H), 3.95 (s, 3H), 3.97(s, 3H), 4.02 (s, 2H), 4.30 (s, 2H), 5.27 (s, 2H), 6.85 (s, 1H), 7.15(dd, 1H, J=7.5, 5.0 Hz), 7.28 (t, 1H, J=7.5 Hz), 7.33 (d, 1H, J=7.5 Hz),7.42 (d, 1H, J=7.5 Hz), 7.47 (d, 1H, J=7.5 Hz), 7.64 (m, 2H), 8.55 (d,1H, J=5.0 Hz).

Example 27 Production of2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F20)

2-(4,5-dimethoxy-2-methoymethoxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F20) was prepared in a similar manner for the preparation of2-(5-methoxy-2-methoxymethoxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F17) using4-bromomethyl-2-(4,5-dimethoxy-2-methoxymethoxyphenyl)benzoxazole (F13)(408 mg, 1.0 mmol) instead of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) in71% (317 mg); ¹H-NMR (500 MHz, CDCl₃) δ2.35 (s, 3H), 3.58 (s, 3H), 3.84(s, 2H), 3.96 (s, 3H), 3.98 (s, 3H), 4.13 (s, 2H), 5.28 (s, 2H), 6.85(s, 1H), 7.15 (dd, 1H, J=7.5, 5.0 Hz), 7.32 (t, 1H, J=8.0 Hz), 7.46–7.50(m, 2H), 7.61–7.69 (m, 3H), 8.55 (d, 1H, J=5.0 Hz).

Example 28 Production of4-bis(2-pyridylmethyl)aminomethyl-2-(4-methoxymethoxyphenyl)benzoxazole(F21)

4-bis(2-pyridylmethyl)aminomethyl-2-(4-methoxymethoxyphenyl)benzoxazole(F21) was prepared in a similar manner for the preparation of4-bis(2-pyridylmethyl)aminomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole(F18) using 4-bromomethyl-2-(4-methoxymethoxyphenyl)benzoxazole (F15)(552 mg, 1.5 mmol) instead of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) in98% (704 mg); ¹H-NMR (400 MHz, CDCl₃) δ3.52 (s, 3H), 3.93 (s, 4H), 4.19(s, 2H), 5.26 (s, 2H), 7.11–7.18 (m, 4H), 7.29 (t, 1H, J=8.0 Hz), 7.44(d, 1H, J=8.0 Hz), 7.51 (d, 1H, J=8.0 Hz), 7.66 (d, 2H, J=7.6 Hz), 7.78(d, 2H, J=7.6 Hz), 8.19 (d, 2H, J=9.2 Hz), 8.55 (d, 2H, J=5.0 Hz).

Example 29 Production of4-bis(2-pyridylmethyl)aminomethyl-2-(2,5-dimethoxyphenyl)benzoxazole(F22)

4-bis(2-pyridylmethyl)aminomethyl-2-(2,5-dimethoxy phenyl)benzoxazole(F22) was prepared in a similar manner for the preparation of4-bis(2-pyridylmethyl)aminomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole(F18) using 4-bromomethyl-2-(2,5-dimethoxyphenyl)benzoxazole (F14) (522mg, 1.5 mmol) instead of4-bromomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole (F12) in91% (640 mg); ¹H-NMR (400 MHz, CDCl₃) δ3.88 (s, 4H), 3.95 (s, 6H), 4.23(s, 2H), 7.01–7.07 (m, 2H), 7.13 (dd, 2H, J=8.0, 5.0 Hz), 7.34 (t, 1H,J=7.5 Hz), 7.49 (d, 1H, J=7.5 Hz), 7.57 (d, 1H, J=7.5 Hz), 7.64–7.68 (m,3H), 7.80 (d, 2H, J=8.0 Hz), 8.52 (d, 2H, J=5.0 Hz).

Example 30 Production of2-(5-methoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethyl-benzoxazole(F23)

A mixture of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethyl-benzoxazole(F16) (283 mg, 0.70 mmol) and p-toluenesulfonic acid monohydrate (532mg, 2.8 mmol) in methanol (20 mL) was stirred overnight at roomtemperature. After the solvent was removed by evaporation, ethyl acetate(20 mL) was added to the resulting residue, neutralized by Na₂CO₃,washed with brine and water, dried over MgSO₄, and concentrated invacuo. The residue was recrystallized from hot ethanol/hexane to affordthe objective compound as a pale yellow crystal (244 mg, 96%); ¹H-NMR(500 MHz, CDCl₃) δ3.86 (s, 3H), 4.01 (s, 2H), 4.21 (s, 2H), 7.05 (br s,2H), 7.17 (dd, 1H, J=7.5, 4.5 Hz), 7.33–7.37 (m, 2H), 7.41 (d, 1H, J=7.5Hz), 7.48–7.51 (m, 2H), 7.66 (t, 1H, J=7.5 Hz), 8.59 (d, 1H, J=4.5 Hz),11.00 (s, 1H).

Example 31 Production of2-(5-methoxy-2-hydroxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F24)

2-(5-methoxy-2-hydroxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethyl-benzoxazole(F24) was prepared in a similar manner for the preparation of2-(5-methoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethyl-benzoxazole(F23) using2-(5-methoxy-2-methoxymethoxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F17) (209 mg, 0.5 mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethyl-benzoxazole(16) in 93% (173 mg); ¹H-NMR (500 MHz, CDCl₃) δ2.32 (s, 3H), 3.81 (s,2H), 3.87 (s, 3H), 4.01 (s, 2H), 7.05 (s, 2H), 7.17 (dd, 1H, J=7.5, 4.5Hz), 7.34 (t, 1H, J=7.5 Hz), 7.46–7.52 (m, 3H), 7.58 (d, 1H, J=7.5 Hz),7.71 (t, 1H, J=7.5 Hz), 8.56 (d, 1H, J=4.5 Hz), 11.13 (s, 1H).

Example 32 Production of4-bis(2-pyridylmethyl)aminomethyl-2-(2-hydroxy-5-methoxyphenyl)benzoxazole(F25)

4-bis(2-pyridylmethyl)aminomethyl-2-(2-hydroxy-5-methoxyphenyl)benzoxazole(F25) was prepared in a similar manner for the preparation of2-(5-methoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F23) using4-bis(2-pyridylmethyl)aminomethyl-2-(5-methoxy-2-methoxymethoxyphenyl)benzoxazole(F17) (136 mg, 0.275 mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F16) in 98% (122 mg); ¹H-NMR (500 MHz, CDCl₃) δ3.87 (s, 3H), 3.89 (s,4H), 4.12 (s, 2H), 7.06 (br s, 2H), 7.14 (dd, 1H, J=7.5, 4.0 Hz), 7.35(t, 1H, J=8.0 Hz), 7.49–7.53 (m, 3H), 7.64–7.70 (m, 4H), 8.52 (d, 2H,J=4.0 Hz), 11.06 (s, 1H).

Example 33 Production of2-(4,5-dimethoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F26)

2-(4,5-dimethoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethyl-benzoxazole(F26) was prepared in a similar manner for the preparation of2-(5-methoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F23) using2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F17) (100 mg, 0.23 mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F16) in 92% (83 mg); ¹H-NMR (500 MHz, CDCl₃) δ3.95 (s, 3H), 3.96 (s,3H), 4.01 (s, 2H), 4.21 (s, 2H), 6.66 (s, 1H), 7.17 (dd, 1H, J=7.5, 5.0Hz), 7.31 (t, 1H, J=8.0 Hz), 7.37–7.40 (m, 2H), 7.41 (s, 1H), 7.48 (d,1H, J=8.0 Hz),7.67 (t, 1H, J=7.5 Hz), 8.66 (d, 2H, J=5.0 Hz), 11.27 (s,1H).

Example 34 Production of2-(4,5-dimethoxy-2-hydroxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F27)

2-(4,5-dimethoxy-2-hydroxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F27) was prepared in a similar manner for the preparation of2-(5-methoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F23) using2-(4,5-dimethoxy-2-methoxymethoxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethylbenzoxazole(F20) (270 mg, 0.6 mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F16) in 94% (221 mg); ¹H-NMR (500 MHz, CDCl₃) δ2.33 (s, 3H), 3.81 (s,2H), 3.95 (s, 3H), 3.96 (s, 3H), 3.99 (s, 2H), 6.65 (s, 1H), 7.17 (dd,1H, J=7.5, 4.5 Hz), 7.31 (t, 1H, J=8.0 Hz), 7.41 (s, 1H), 7.44–7.48 (m,2H), 7.59 (d, 1H, J=8.0 Hz), 7.71 (t, 1H, J=7.5 Hz), 8.56 (d, 2H, J=4.5Hz), 11.39 (s, 1H).

Example 35 Production of4-bis(2-pyridylmethyl)aminomethyl-2-(4-hydroxyphenyl)benzoxazole (F28)

4-bis(2-pyridylmethyl)aminomethyl-2-(4-hydroxyphenyl)benzoxazole (F28)was prepared in a similar manner for the preparation of2-(5-methoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F23) using4-bis(2-pyridylmethyl)aminomethyl-2-(4-methoxymethoxyphenyl)benzoxazole(F21) (686 mg, 1.47 mmol) instead of2-(5-methoxy-2-methoxymethoxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole(F16) in 81% (500 mg); ¹H-NMR (500 MHz, CDCl₃) δ3.97 (s, 4H), 4.18 (s,2H), 6.88 (d, 2H, J=9.0 Hz), 7.16 (dd, 2H, J=8.0, 4.5 Hz), 7.24 (t, 1H,J=8.0 Hz), 7.40 (d, 1H, J=8.0 Hz), 7.45 (d, 2H, J=8.0 Hz), 7.69 (t, 2H,J=8.0 Hz), 7.79 (d, 2H, J=8.0 Hz), 8.00 (d, 2H, J=9.0 Hz), 7.24 (d, 2H,J=4.5 Hz).

Example 36

Production of zinc complex using Formula 25. To a solution of4-bis(2-pyridylmethyl)aminomethyl-2-(2-hydroxy-5-methoxyphenyl)benzoxazole(F25) (133 mg, 0.27 mmol) and triethylamine (38 μL, 0.27 mmol) inmethanol (5 mL) was added ZnCl₂ (36.5 mg, 0.27 mmol), and the mixturewas stirred for 2 h. KPF₆ (184 mg, 1.0 mmol) was then added into thesolution and the mixture was stirred for an additional 2 h. Theresulting solution was concentrated in vacuo, and the residue wasdissolved in small amount of methanol that was poured into ether (50mL). The precipitate was collected by filtration and washed with waterto afford yellow powder (135 mg, 75%). Single crystals (yellow cubic)for X-ray structure determination were obtained by recrystallizationfrom methanol/ether diffusion.

Example 37

Production of zinc complex using Formula 24. To a solution of2-(5-methoxy-2-hydroxyphenyl)-4-N-(2-pyridylmethyl)-N-methyl-aminomethyl-benzoxazole(F24) (75 mg, 0.2 mmol) and triethylamine (28 μL, 0.2 mmol) in methanol(5 mL) was added ZnCl₂ (27 mg, 0.2 mmol), and the mixture was stirredfor 3 h. The reaction mixture was subsequently poured into ether (50 mL)and the precipitate was collected by filtration (385 mg, 81%). Singlecrystals (yellow cubic) for X-ray structure determination were obtainedby recrystallization from methanol/ether diffusion.

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood that thesedescriptions are added only by way of example and are not intended tolimit, in any way, the scope of this invention. For instance, compoundsof the present invention can be extended to include a range of analogousfused heterocyclic compounds (e.g., benzthiazoles, etc.) usingstraightforward modifications of the synthetic techniques describedherein, such techniques as would be understood by those skilled in theart made aware of this invention. Likewise, substitution at the4-position of the fused heterocyclic system can vary, limited only bydesired interaction with Zn⁺² metal ion and/or solvent solubility.Regardless of the compound or substitution, various imaging orspectroscopic techniques, known in the art, can be used in conjunctiontherewith.

1. A fluorescent compound of a formula

wherein R₁ and R₂ are independently selected from hydrogen, hydroxy,alkyl and alkoxy; and X₁ is (2-pyridinylalkyl); and X₂ is selected fromhydrogen, alkyl, and alkylhydroxy.
 2. The compound of claim 1 wherein X₂is selected from alkyl and hydrogen.
 3. The compound of claim 1 whereinX₁ is (2-pyridinylmethyl) and X₂ is methyl.
 4. The compound of claim 1wherein X₁ is (2-pyridinylmethyl) and X₂ is hydrogen.